Amidated glucagon-like peptide-1

ABSTRACT

The present invention encompasses a GLP-1 analog and compositions and formulations thereof useful for the treatment of hyperglycemia and other various diseases and conditions in mammals.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/255,251, filed Dec. 13, 2000.

[0002] Glucagon-Like Peptide 1 (GLP-1) is a 37 amino acid peptide thatis secreted by the L-cells of the intestine in response to foodingestion. It has been found to stimulate insulin secretion(insulinotropic action), thereby causing glucose uptake by cells anddecreased serum glucose levels (see, e g., Mojsov, S., Int. J. PeptideProtein Research, 40:333-343 (1992)). These and other effects make GLP-1an attractive candidate for the treatment of type 2 diabetes.

[0003] Thus far, however, efforts to commercially develop a GLP-1 drugcandidate have been hampered by numerous obstacles. For example, thedevelopment of native GLP-1 compounds has not been feasible because theyare rapidly degraded by endogenous proteases and thus, have an extremelyshort in vivo half-life. Although analogs with longer half-lives havebeen studied, these analogs have been difficult to develop commerciallydue to stability problems encountered during the manufacturing process.

[0004] One particular analog that has been studied isVal⁸-GLP-1(7-37)OH. See U.S. Pat. No. 5,977,071. This analog has apotency similar to that of the truncated native GLP-1 peptidesGLP-1(7-36)NH₂ and GLP-1(7-37)OH but has a longer circulating half-life.Val⁸-GLP-1(7-37)OH, however, exists in at least two different forms. Thefirst form is physiologically active and dissolves readily in aqueoussolution whereas the second form is substantially insoluble in water atphysiological pH and is inactive. Further, Val⁸-GLP-1(7-37)OH has atendency to aggregate and convert to an inactive insoluble form duringmanufacturing. Thus, efforts to improve the properties of this compoundhave continued. Discovery efforts have focused on improving thestability of the compound in the context of large scale manufacturing aswell as formulation development without compromising the biologicalactivity.

[0005] The present invention encompasses a Val⁸-GLP-1 analog having suchimproved properties. The invention encompasses the discovery that anamidated form of Val⁸-GLP-1(7-37)OH known as Val8-GLP-1(7-37)NH₂ hasincreased in vitro potency compared to Val⁸-GLP-1(7-37)OH (the acidform) and has superior stability properties that facilitate large-scalemanufacturing and make it an ideal candidate to formulate as a solutionfor continuous infusion or as a crystal suspension for subcutaneousadministration.

[0006] Although native GLP-1 is amidated in vivo, there has been nomotivation to study amidated analogs because both native isoforms appearto have identical biological effects and amidated peptides are perceivedas more difficult to make biosynthetically. In addition, unlike theamidated analog encompassed by the present invention, amidation of thenative molecule actually involves replacement of the C-terminalglycine-OH with NH2 resulting in GLP-1(7-36)NH₂. Further, thecorresponding Val⁸-GLP-1(7-36)NH₂ analog, disclosed in U.S. Pat. No.6,133,235, actually turns out to be slightly less potent than the acidform.

[0007] Thus, it is surprising that Val⁸-GLP-1(7-37)NH₂ has increasedstability as a formulated compound as well as increased stability in thecontext of manufacturing processes over the acid form of the analog.Even more surprising is the increased potency compared to the acid formor the truncated amide form of the analog.

[0008] The present invention comprises a peptide having the sequencerepresented in Formula I (SEQ ID NO:1). Formula I (SEQ ID NO: 1)7   8   9   10  11  12  13  14  15  16  17His-Xaa-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-18  19  20  21  22  23  24  25  26  27  28Ser-Tyr-Leu-Glu-Xaa-Gln-Ala-Ala-Lys-Xaa-Phe-29  30  31  32  33  34  35  36  37 Ile-Xaa-Trp-Leu-Val-Lys-Gly-Arg-R

[0009] wherein:

[0010] Xaa at position 8 is Val; Xaa at position 22 is Gly;

[0011] Xaa at position 27 is Glu; Xaa at position 30 is Ala;

[0012] and R is Gly-NH₂;

[0013] The present invention also comprises a solution formulation ofthe peptide corresponding to SEQ ID NO:1. The present invention furthercomprises crystals of the peptide corresponding to SEQ ID NO:1 as wellas pharmaceutical compositions of such crystals.

[0014] The three-letter abbreviation code for amino acids used in thisspecification conforms with the list contained in Table 3 of Annex C,Appendix 2 of the PCT Administrative Instructions and with 37 CFR §1.822(d)(1)(2000).

[0015] It has now been found that Val8-GLP-1(7-37)NH₂ shows a markedlydecreased propensity to aggregate in solution compared withVal⁸-GLP-1(7-37)OH yet can still be crystallized to provide extendedtime action similar to Val⁸-GLP-1(7-37)OH. Further, Val8-GLP-1(7-37)NH₂has an in vitro potency that is approximately 1.2-fold greater thanVal⁸-GLP-1(7-37)OH.

[0016] As accustomed in the art, the N-terminal residue of a GLP-1related peptide is represented as position 7. Thus, the amino acidsequence of the naturally occurring human GLP-1 related peptidedesignated GLP-1(7-37)OH is: (SEQ ID NO: 2)7   8   9   10  11  12  13  14  15  16  17His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-18  19  20  21  22  23  24  25  26  27  28Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-29  30  31  32  33  34  35  36  37 Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly

[0017] Val⁸-GLP-1(7-37)NH₂ is GLP-1(7-37)OH wherein Ala at position 8has been substituted with Val and Gly at position 37 has been amidated.

[0018] The amino acid sequence of Val⁸-GLP-1(7-37)OH is: (SEQ ID NO: 3)7   8   9   10  11  12  13  14  15  16  17His-Val-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-18  19  20  21  22  23  24  25  26  27  28Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-29  30  31  32  33  34  35  36  37 Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly

[0019] The word “crystal” in the present specification refers to a solidmaterial comprising a peptide in which the particles making up the solidhave a definite form or structure. Particles lacking such form orstructure are referred to as “amorphous.” Words used in the presentspecification to describe crystals include, in order of increasingcrystal quality; 1) “microcrystals,” which are small crystals possessinga definite but essentially non-linear form or structure, 2) “stars” or“clusters,” which are distinct crystals fused at or emanating from acentral core and which may contain amorphous material in addition tocrystalline material, and 3) “rods,” “needles,” or “plates” which areindividual crystals possessing a distinctive form or structureconsistent with their name.

[0020] The term “thin plate crystals” refers to individual peptidecrystals having an apparent orthorhombic structure in which the threeaxes of the crystals have disparate lengths. The thin plate crystals ofthe present invention generally have a thickness of about 0.5 μm toabout 3.0 μm, a width of about 3 μm to about 10 μm and a length of about10 μm to about 100 μm. Under the microscope thin plate crystals mayappear orthorhombic but the actual angles between the axes may or maynot be 90°.

[0021] The word “stable” used in the present specification refers to acomposition in which both the chemical stability and physical stabilityof the composition remain at an acceptable level over time. The word“chemical” used in conjunction with stability of a peptide compositionrefers to covalent modifications or alterations of the peptide. The word“physical” used in conjunction with stability of a peptide compositionrefers to the molecular conformation, solubility or solid formproperties of the peptide. By way of illustration, peptide crystalcompositions in which the crystals quickly clump into large aggregatesor dissolve into the liquid medium exhibit unsatisfactory physicalstability.

[0022] The word “pharmaceutical” used in the present specification inreference to a peptide composition means it contains a peptide usefulfor treating a disease or condition. For example, the peptideVal⁸-GLP-1(7-37)NH₂ described in the present invention is useful intreating humans and other mammals who have Type 2 diabetes.

[0023] The word “treating” refers to the management and care of apatient for the purpose of combating a disease, condition, or disorderand includes the administration of crystals, a pharmaceuticalcomposition thereof, or a pharmaceutically-acceptable solutionformulation to prevent the onset of the symptoms or complications,alleviating the symptoms or complications, or eliminating the disease,condition or disorder. Treating diabetes therefore includes themaintenance of physiologically desirable blood glucose levels inpatients in need thereof.

[0024] For the convenience and safety of patients being treated,pharmaceutical compositions of the present invention also contain apharmaceutically acceptable preservative. A “pharmaceutically acceptablepreservative” refers to a chemical that is compatible with and suitablefor pharmaceutical use in humans and that is added to a peptidecomposition to prevent or inhibit the growth of micro-organisms. Theterm “phenolic preservative” as used herein refers to a pharmaceuticallyacceptable preservative containing a phenolic moiety and includesphenol, m-cresol, methylparaben and mixtures thereof. Utilizing apharmaceutically acceptable preservative in a peptide composition allowsa patient to conveniently make multiple withdrawals of the compositionfrom the same container, such as a vial or cartridge, over an extendedperiod of time.

[0025] The word “buffer” refers to a chemical compound in a compositionthat minimizes changes in hydrogen ion concentration that wouldotherwise occur as a result of a chemical reaction and includes, but isnot limited to, TRIS, maleate, phosphate, succinate, glycylglycine,adipate, citrate, and acetate.

[0026] The term “TRIS” refers to2-amino-2-hydroxymethyl-1,3-propanediol, and any pharmaceuticallyacceptable salt thereof. The free base and the hydrochloride form aretwo common forms of TRIS. TRIS is also known in the art as trimethylolaminomethane, tromethamine and tris(hydroxymethyl)aminomethane.

[0027] The word “maleate” refers to maleic acid, which has the chemicalformula HOOCCH:CHCOOH, and pharmaceutically acceptable salt forms suchas sodium maleate and potassium maleate.

[0028] The word “succinate” refers to succinic acid, which has thechemical formula CO₂H(CH₂)₂CO₂H, and pharmaceutically acceptable saltforms such as sodium succinate and potassium succinate.

[0029] The word “adipate” refers to adipic acid, which has the chemicalformula CO₂H(CH₂)₄CO₂H, and pharmaceutically acceptable salt forms suchas sodium adipate and potassium adipate.

[0030] The word “glycylglycine” refers to the dipeptide Gly-Gly, thefree base form of Gly-Gly and pharmaceutically acceptable salt formssuch as glycylglycine hydrochloride.

[0031] The word “ethanol” is synonymous with ethyl alcohol and refers tothe chemical CH₃CH₂OH.

[0032] The word “isopropanol” is synonymous with isopropyl alcohol andrefers to the chemical (CH₃)₂CHOH.

[0033] The term “tonicity agent” refers to a non-volatile chemicalcompound that modifies the osmotic pressure of a solution or suspensioncomposition and includes sodium chloride, other salts, glycerin andmannitol.

[0034] “TCR” stands for “Temperature Cycling and Resuspension” andrefers to the automated test of pharmaceutical compositions involvingtemperature cycling and physical resuspension described herein as Method2. The “Modified TCR Test” refers to the test described herein as Method3.

[0035] The symbol “%” is equivalent to the word “percent” and, as usedherein in reference to a volume of a specified liquid within a largerliquid composition or added to a liquid composition, means the actualvolume of the specified liquid divided by the total volume of thecombined composition after the specified liquid is added, multiplied by100.

[0036] As noted, the present invention comprises a peptide, known asVal⁸-GLP-1(7-37)NH₂, having the sequence represented in formula I (SEQID NO:1). Formula I (SEQ ID NO: 1)7   8   9   10  11  12  13  14  15  16  17His-Xaa-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-18  19  20  21  22  23  24  25  26  27  28Ser-Tyr-Leu-Glu-Xaa-Gln-Ala-Ala-Lys-Xaa-Phe-29  30  31  32  33  34  35  36  37 Ile-Xaa-Trp-Leu-Val-Lys-Gly-Arg-R

[0037] wherein:

[0038] Xaa at position 8 is Val; Xaa at position 22 is Gly;

[0039] Xaa at position 27 is Glu; Xaa at position 30 is Ala;

[0040] and R is Gly-NH₂;

[0041] Val⁸-GLP-1(7-37)NH₂ may be prepared by various methods. Forexample, the peptide may be chemically synthesized by solution-phase,solid-phase or semi-synthetic methods (see Example 1). Alternatively,the peptide may be prepared biosynthetically using recombinant DNAtechnology in modified bacteria, yeast, mammalian cells or in transgenicplants or animals and then amidated using a transpeptidation reaction(see Example 2).

[0042] Transpeptidation is a reaction that uses a proteolytic enzyme toboth cleave a peptide bond to remove an amino acid residue(s) from asubstrate peptide and subsequently form a new peptide bond between thesubstrate and la new amino acid residue. Thus, Val⁸-GLP-1(7-37)OH can beprepared biosynthetically and then converted to Val⁸-GLP-1(7-37)NH₂ byremoving the C-terminal glycine and replacing it with anamidated-glycine (Gly-NH₂) residue. This can be achieved by adding watermiscible organic solvents to the reaction mixture and having a largemolar excess of the amidated amino acid residue in solution. Forexample, Val⁸-GLP-1(7-37)OH can be converted to Val⁸-GLP-1(7-37)NH₂using trypsin-mediated or carboxypeptidase Y-mediated transpeptidation.Trypsin is preferred because of its selectivity for basic amino acidresidues such as lysine and arginine and the penultimate amino acidresidue at the C-terminal end of Val⁸-GLP-1(7-37)OH is arginine. Toprevent trypsin from digesting Val⁸-GLP-1(7-37)OH at each of the twointernal lysines, these lysines can be modified to prevent digestion.For example, the lysines can be acylated using citriconic anhydride inan aqueous buffered solution to temporarily protect the lysine residuesfrom trypsin digestion. Further this type of a reaction caps theN-terminal amino group of the peptide. Once the protectedVal⁸-GLP-1(7-37)OH molecule is treated with trypsin, the acyl groups canbe removed.

[0043] The Val⁸-GLP-1(7-37)NH₂ peptide has slightly increased in vitroactivity compared to Val⁸-GLP-1(7-37)OH and has a substantially reducedtendency to aggregate in solution. Val⁸-GLP-1(7-37)NH₂ andVal⁸-GLP-1(7-37)OH were analyzed with respect to their potential toaggregate in solution (see Example 3). The peptides in solution werestirred at elevated temperature in a suitable buffer while recordingturbidity at 350 nm as a function of time. Time to the onset ofaggregation was measured to quantify the potential of each GLP-1molecule to aggregate under these stressed conditions.Val⁸-GLP-1(7-37)NH₂ had an aggregation time of approximately 23 hours at37° C. compared to 1 hour for Val⁸-GLP-1(7-37)OH at 37° C. Thus,Val⁸-GLP-1(7-37)NH₂ is less likely to aggregate and convert to aninactive insoluble form during manufacturing and will have superiorstability as a formulated solution or crystal suspension.

[0044] The present invention also encompasses apharmaceutically-acceptable solution formulation and a lyophilizedformulation that can be reconstituted as a solution comprisingVal⁸-GLP-1(7-37)NH₂. These types of formulations are most useful forcontinuous intravenous (i.v.) infusion of Val⁸-GLP-1(7-37)NH₂.Continuous infusion means continual and substantially uninterruptedadministration of a solution for a specified period of time. Suchformulations are also useful when a bolus injection is desired. A bolusinjection is the injection of a drug in a defined quantity at once. Dueto the relatively short half-life of Val⁸-GLP-1(7-37)NH₂ whenadministered as a solution, it is preferred that a solution of thismolecule be administered by continuous infusion.

[0045] It is known that GLP-1 molecules have effects beyond glucosenormalization in diabetics. For example, GLP-1 has been shown to reducethe mortality and morbidity associated with stroke and myocardialinfarction as well as effect catabolic changes that occur after surgery.See WO 98/08531, WO 98/08873, and WO 00/16797. Post-surgical patients aswell as patients who have suffered an MI or stroke generally spend sometime in the hospital. Thus, a solution formulation or a lyophilizedformulation that could be reconstituted is particularly suitable fori.v. administration in a hospital setting.

[0046] A solution formulation is comprised of Val⁸-GLP-1(7-37)NH₂dissolved in an aqueous solution. Such formulation has the appropriatestability and is suitable for intravenous administration. Preferably,the solution formulation is comprised of a pharmaceutically-acceptablebuffer, and the pH adjusted to maintain solubility and provideacceptable stability. Optionally, one or morepharmaceutically-acceptable preservatives such as a phenolicpreservative may be added. Meta-cresol and phenol are preferredpharmaceutically-acceptable preservatives. One or morepharmaceutically-acceptable salts may also be added to adjust the ionicstrength or tonicity. One or more excipients may be added to furtheradjust the isotonicity of the formulation. Glycerin is an example of anisotonicity-adjusting excipient. One or more excipients may be added tocontrol non-specific adsorption of the protein to the administrationdevice or equipment (e.g., tubing, i.v. bag). Examples of suchexcipients include but is not limited to human serum albumin anddetergents.

[0047] Alternatively, Val⁸-GLP-1(7-37)NH₂ can be prepared as alyophilized product and then reconstituted before administration. Apreferred lyophilized product is comprised of Val⁸-GLP-1(7-37)NH₂ and abulking agent such as mannitol, trehalose, raffinose, and sucrose. Thepreferred concentration of Val⁸-GLP-1(7-37)NH₂ in the lyophilizedproduct is between about 0.5 mg and about 20 mg, preferably betweenabout 1 mg and about 10 mg, most preferably between about 1 mg and about5 mg. Prior to lyophilization and upon reconstitution, it is preferableto maintain the pH range between about 6 and about 10, preferablybetween about 6.5 and about 8 and most preferably between about 7 andabout 7.5.

[0048] Accordingly, upon lyophilization, the product optionallycomprises a pharmaceutically acceptable buffer. Preferred buffer systemsinclude Tris, citrate, phosphate, and maleate based buffers. Thebuffering capacity of the lyophilized product may be used to control thepH of the i.v. solution to maximize stability of the peptide. Forexample, the pH of standard 0.9% NaCl which is unbuffered and has a pHin the range of 4.5 to 7.0 can be raised to a pH range where the peptideis stabilized. Further, upon lyophilization, the product optionallycomprises a salt to generate ionic strength. Representative saltsinclude, but are not limited to, potassium chloride (KCl) and sodiumchloride (NaCl).

[0049] The lyophilized product is reconstituted with the appropriatediluent such as sterile water or sterile saline. Preferably the diluentis compatible with whatever i.v. fluid a patient may be receiving in thehospital such that if injected directly into an i.v. line or i.v. bagalready attached to a patient, Val⁸-GLP-1(7-37)NH₂ does not precipitateor aggregate and remains soluble and stable until delivery to thepatient occurs. Typical i.v. solutions are represented in Table I.Solution Na⁺ K⁺ Ca⁺² Cl⁻ Lactate PO₄ ⁻³ Mg⁺² Normal Saline (NS) 154 0 0154 0 0 0 0.2% NS 31 0 0 31 0 0 0 3% NaCl 513 0 0 513 0 0 0 Ringer'sLactate 130 4 3 109 28 0 0 (LR)

[0050] Infusion rates for the solution formulations of the presentinvention should be such that Val⁸-GLP-1(7-37)NH₂ concentrations in theserum remain in the therapeutic range during a course of treatment.Preferably, not more than a total amount of about 5 mg ofVal⁸-GLP-1(7-37)NH₂ should be administered within a 24 hour period. Evenmore preferably, not more than a total amount between about 1 mg andabout 3 mg should be administered within a 24 hour period. Mostpreferably a total dose of about 1 mg should be administered per 24 hourperiod.

[0051] The present invention also encompasses crystals comprising apeptide of Formula I (SEQ ID NO:1) as well as pharmaceuticalcompositions comprising such crystals. A pharmaceutical composition asused herein is comprised of a suspension of peptide crystals.

[0052] Thin plate crystals described herein have the appearance oforthorhombic crystals in which the three axes have disparate lengths.Although the lengths of the axes are not to be construed as limited tospecific dimensions, the thin plate crystals of the present inventiongenerally have a thickness of about 0.5 μm to about 3.0 μm, a width ofabout 3 μm to about 10 μm and a length of about 10 μm to about 100 μm.Under the microscope thin plate crystals may appear orthorhombic but theactual angles between the axes may or may not be 90°.

[0053] In addition to their surface charge, the shape and dimensions ofthin plate crystals are important properties that can provide a slowsedimentation rate in a pharmaceutical composition of the presentinvention. Preferably, the thickness, width and length of the peptidecrystals are about 0.5-1.5 μm, about 3-8 μm, and about 15-80 μm,respectively. More preferably, the thickness, width and length of thecrystals are about 0.8-1.2 μm, about 4-6 μm and about 20-60 μm,respectively. Most preferably, the thickness, width and length of thecrystals are about 1 μm, about 5 μm and about 30-50 μm, respectively.Analysis of the sedimentation rates and volumes of pharmaceuticalcompositions comprising these peptide crystals is performed as describedin Method 1. Preferably the sedimentation volume after 24 hours isbetween about 53% and about 72% of total volume.

[0054] The pharmaceutical compositions of the present invention comprisecrystals of Val⁸-GLP-1(7-37)NH₂, zinc, an alcohol selected from thegroup consisting of ethanol and isopropanol, a buffer, apharmaceutically acceptable preservative, and a pH between about 6.0 andabout 8.5. Compositions of Val⁸-GLP-1(7-37)NH₂ comprising zinc andalcohol are further stabilized in the presence of glycine and bufferselected from the group consisting of TRIS and maleate. Additionalpharmaceutically acceptable excipients, such as those described inRemington's Pharmaceutical Sciences (1985) may be included in thecompositions of the present invention. Preferably, such additionalexcipients do not affect the novel and basic characteristics of theinvention. Therefore, the chemical and physical stability and thetherapeutic benefit of the pharmaceutical composition are retained.

[0055] Preferably, the total peptide concentration in the compositionsof the present invention is about 1.0 mg/mL to about 50 mg/mL. Morepreferably, the peptide concentration is about 2.0 mg/mL to about 30mg/mL. Other ranges of preferred concentrations of peptide are about 5.0to about 20.0 mg/mL, about 5.0 to about 10.0 mg/mL and about 2.0 mg/mLto about 8.0 mg/mL. A most preferred peptide concentration is about 6.0mg/mL.

[0056] The compositions of the present invention comprise glycine at aconcentration of about 5 mM to about 100 mM. Preferably, the glycineconcentration is about 10 mM to about 50 mM. More preferably, theglycine concentration is about 20 mM to about 30 mM and more highlypreferred is a glycine concentration of about 22 mM to about 24 mM. Aglycine concentration of about 23 mM is most preferred.

[0057] The compositions of the present invention comprise an alcoholpreferably selected from the group consisting of ethanol and isopropanolat a concentration, by total volume of the composition, of about 1% toabout 10%. A preferred concentration of the alcohol in the compositionsis about 2% to about 6% by volume. More preferred is an alcoholconcentration of about 4%. A preferred alcohol is ethanol.

[0058] The compositions of the present invention comprise zinc at aconcentration of about 0.2 moles to about 2.5 moles per mole of thepeptide. The zinc present in the compositions is generally in the formof a zinc ion derived from zinc oxide or from zinc salts such as zincchloride or zinc acetate. A preferred concentration of zinc is about 1.0to about 2.25 moles per mole of the peptide in the composition. Otherranges of preferred zinc concentrations in the compositions are about1.1 moles to about 2.0 moles per mole of the peptide and about 1.3 molesto about 1.7 moles per mole of the peptide. A more preferred zincconcentration is about 1.5 moles per mole of the peptide.

[0059] The compositions of the present invention may comprise a tonicityagent such as sodium chloride. Other tonicity agents, such as glycerin,mannitol and salts other than sodium chloride, may also be incorporatedinto the compositions in addition to or in place of sodium chloride. Thequantities of sodium chloride (NaCl) noted in this specification referto the quantities of sodium chloride added to a composition at adesignated point in the preparation of the composition. The NaClquantities noted in the specification do not include sodium chloridethat may form from additions of acids and bases such as NaOH and HClthat may be used for pH adjustment at various stages in the preparationof the compositions. Also, it is appreciated that when sodium chlorideis added to an aqueous composition a substantial portion will exist assodium ions and chloride ions. For ease of measurement andunderstanding, however, the sodium and chloride ion concentrations ofthe compositions will not be considered, only the quantity of sodiumchloride added.

[0060] When included in a composition of the present invention, apreferred concentration of sodium chloride in the composition is about30 mM to about 200 mM. A more preferred quantity of sodium chloride is50 mM to about 150 mM. Other ranges of preferred sodium chlorideconcentration are about 80 mM to about 120 mM, about 70 mM to about 130mM, and about 90 mM to about 130 mM. A most preferred concentration ofsodium chloride in the compositions of the present invention is about110 mM.

[0061] The compositions of the present invention may also comprise abuffer. Preferably, the buffer is selected from the group consisting ofTRIS and maleate or combinations thereof.

[0062] A preferred range of concentration of TRIS in the compositions ofthe present invention, if TRIS is the selected buffer, is about 5 mM toabout 40 mM. A more preferred range of concentration of TRIS is about 10mM to about 20 mM. A most preferred concentration of TRIS is about 15mM.

[0063] A preferred range of concentration of maleate in the compositionsof the present invention, if maleate is the selected buffer, is about 2mM to about 20 mM. A more preferred range of concentration of maleate isabout 5 mM to about 15 mM. A most preferred concentration of maleate isabout 9 mM.

[0064] The compositions of the present invention comprise a hydrogen ionconcentration, or pH, which is about 6.0 to about 8.5. The preferred pHrange of the pharmaceutical compositions will also depend to some extentupon the selected peptide and the selected buffer. With TRIS as thebuffer, a preferred range of composition pH is about 6.5 to about 8.5.More preferred ranges of pH are about 7.0 to about 7.8, about 7.2 toabout 7.8, about 7.5 to about 8.5, and about 7.0 to about 8.0. With TRISas the buffer, a most preferred pH is about 7.5. With maleate as thebuffer, a preferred range of composition pH is about 6.0 to about 7.5.More preferred ranges of pH are about 6.4 to about 7.5, about 6.4 toabout 7.0, and about 6.0 to about 7.0. With maleate as the buffer, amost preferred pH is about 6.5.

[0065] The compositions of the present invention comprise apharmaceutically acceptable preservative. Preservatives provide safetyand convenience to patients using pharmaceutical compositions.Antimicrobial agents may be added to a product formulation to protectthe product from accidental microbial contamination during itsmanufacture, shelf life and use. This protection is also important whenvials or cartridges containing a composition are provided that allowmultiple withdrawls of the product. Selection and efficacy ofpharmaceutically acceptable preservatives may also be guided by nationalregulatory agencies.

[0066] For the compositions of the present invention, pharmaceuticallyacceptable phenolic preservatives and benzyl alcohol are preferred.Examples of such phenolic preservatives include phenol, chlorocresol,m-cresol, o-cresol, p-cresol, ethylparaben, methylparaben,propylparaben, butylparaben and thymol, and mixtures thereof. Morepreferred preservatives are benzyl alcohol, m-cresol, phenol,methylparaben and mixtures thereof. A most preferred preservative ism-cresol.

[0067] A preferred concentration of preservative in the compositions ofthe present invention is about 1.0 mg/mL to about 20.0 mg/mL. Ranges ofmore preferred concentrations of preservative are about 2.0 mg/mL toabout 8.0 mg/mL, about 2.5 mg/mL to about 4.5 mg/mL and about 2.0 mg/mLto about 4.0 mg/mL. A most preferred concentration of preservative isabout 3.0 mg/mL.

[0068] The pharmaceutical compositions of the present invention aresuitable for use as a medicament for treating diabetes, hyperglycemia,obesity, or related conditions in mammals.

[0069] In order to provide safety, convenience and precise dosing inadministering a suspension composition, suspended material should settlevery slowly in the liquid composition, it should not compact tightly atthe bottom of the container upon storage and settled material shouldresuspend readily with minimal swirling or agitation. For example, it ispreferable that the sedimentation volume be greater than 50% after 24hours of settling at ambient temperature.

[0070] In order to provide accurate dosing and safety to patientsadministering a suspension composition, the suspended material of thecomposition should not agglomerate or clump irreversibly after normalpatient use and storage for a period of time as specified by the productlabel. Such agglomerated or clumped material may clog the orifice of asyringe needle or other device used to administer the composition,thereby reducing the quantity of peptide delivered. Preferably, thecrystal compositions of the present invention, by visual examination,exhibit satisfactory physical stability for at least 14 days in the TCRTest and for at least 28 days in the Modified TCR Test. The TCR Testespecially exaggerates storage and agitation conditions beyond thatexpected by normal patient use. Preferably, the crystal particles in thecompositions of the present invention maintain a small size, that is,about 5 μm to about 10 μm (mean volume percent distribution), for atleast 28 days in the Modified TCR Test.

[0071] Chemical degradation may also occur in peptide compositionsleading to formation of compounds which have reduced potency and/orunknown side effects. Thus, in order to provide safety and full dosingto patients administering peptide pharmaceutical compositions, the levelof chemical degradation occurring in the composition during patient useand storage should be kept to a minimum. It is preferable that HPLCevaluations of peptide compositions that undergo the 14 day TCR or 28day Modified TCR Tests show less than about 2% of the peptide aschemically altered.

[0072] The optimal clinical benefits of the crystal peptide compositionsof the present invention are realized when reproducible, prolongedabsorption is achieved. As a corollary to this goal, adverse eventsresulting from dose dumping that may occur soon after administration ofa composition may be avoided if the quantity of soluble peptide in thecomposition, which tends to be absorbed very quickly, is minimizedduring the typical use and storage conditions of the patient. It ispreferable that the concentration of soluble peptide in the compositionsof the present invention are 8 μg/mL or less after 14 days in the TCRTest or after 28 days in the Modified TCR Test. This means that lessthan 0.3% of the peptide becomes solubilized during the course of thesetests.

[0073] For pharmaceutical compositions of peptide crystals, anotheraspect of physical stability is maintenance of the proper molecularconformation of the peptide. This proper conformation may be critical todelivering a molecule capable of interacting with its receptor andeliciting the desired biological response. A predominantly α-helixconformation is believed to be important in providing a soluble and,therefore, bioavailable peptide while a mostly β-sheet form is believedto be essentially insoluble and, therefore, not bioavailable. Theconformation of Val⁸-GLP-1(7-37)NH₂ in the pharmaceutical compositionsof the present invention can be evaluated by FTIR analysis after 14 daysin the TCR Test. The peptide should maintain a predominantly α-helixconformation throughout the 14-day test. Therefore, the peptide in thesecompositions will be maximally bioavailable after administration to amammal.

[0074] The clinical benefits of the crystal compositions of the presentinvention are realized when the administered peptide is present in themammal being treated for a prolonged period of time.

[0075] The crystals of Val⁸-GLP-1(7-37)NH₂ and compositions thereofaccording to the present invention may be used as a medicament or inpreparing a medicament for the treatment of diabetes, hyperglycemia,obesity, irritable bowel syndrome or related conditions in mammals suchas humans. The present invention also provides a method of treatingdiabetes, hyperglycemia, obesity, irritable bowel syndrome or relatedconditions in mammals such as humans, which comprises administering tothe mammal crystals of a selected peptide or a pharmaceuticalcomposition thereof.

[0076] A dose of 0.001 to 5 mg of a peptide in crystals or compositionsthereof per kg of body weight of a mammal may be administeredparenterally to the mammal in need of such treatment. One skilled in theart will recognize that smaller or larger doses may also be operable,depending on the patient, their condition and the manner ofadministration. Preferred dose ranges include 0.001 to 1 mg/kg, 0.002 to3 mg/kg, 0.005 to 2 mg/kg, 0.01 to 1 mg/kg, 0.01 to 0.1 mg/kg, 0.2 to0.8 mg/kg, 0.8 to 3 mg/kg and 0.2 to 3 mg/kg. More preferred dose rangesinclude 0.001 to 1 mg/kg, 0.005 to 2 mg/kg,-0.01 to 1 mg/kg, and 0.01 to0.1 mg/kg.

[0077] A total dose of 0.01 to 20 mg, based on the mass of the crystalsor the mass of the crystals in the compositions of the presentinvention, may be administered parenterally to a mammal, such as ahuman, in need of such treatment. One skilled in the art will recognizethat a smaller or larger total dose may also be operable, depending onthe patient, their condition and the manner of administration. Preferredtotal dose ranges include 0.01 mg to 10 mg, 0.1 to 10 mg, 1 to 8 mg and2 to 6 mg. More preferred total dose ranges are 1 to 8 mg and 2 to 6 mg.

[0078] Pharmaceutical compositions of the present invention may beadministered parenterally to patients in need thereof more than once perday, once per day, once every two days, twice per week, once per week,or in other dosing regimens known to those skilled in the art. Apreferred dosing regimen is administration once per day.

[0079] The claimed compositions may be administered to a patient in needthereof by a variety of parenteral delivery methods appreciated by theskilled artisan. Preferred methods include subcutaneous injection,intramuscular injection, pulmonary administration, buccal, nasal, ortransdermal delivery, and delivery by internal or external pump. Morepreferred delivery methods are subcutaneous and intramuscular injection.When injected, syringes or cartridges employing needles or needle-lessdevices well known in the art may be employed.

[0080] The compositions of the present invention are preferably preparedas described herein. The first step in this process is dissolving aselected peptide in a glycine-free solution at a pH of about 9.5 toabout 11.5. This “alkaline normalization” step appears to reduce thecontent of β-sheet conformation in the peptide and enhance the α-helixconformation that is important for solubility and bioavailability. Thisstep also serves to maintain the peptide in a preferred α-helixconformation prior to the subsequent process step. This key step thus“normalizes” variation in bulk lots of the peptide into a morereproducible, homogenous solution.

[0081] Preferably, the peptide concentration in the alkalinenormalization solution is greater than 5 mg/mL. More preferably, thepeptide concentration is about 10 mg/mL to about 30 mg/mL. Other rangesof preferred concentration of dissolved peptide are about 5 mg/mL toabout 25 mg/mL, about 8 mg/mL to about 25 mg/mL and about 10 mg/mL toabout 20 mg/mL. The most preferred peptide concentration is about 15mg/mL.

[0082] Preferably, an aqueous alkaline solution comprising only waterand a base such as NaOH, KOH or ammonium hydroxide is employed todissolve the peptide. A more preferred base is NaOH.

[0083] Preferably, the pH of the alkaline normalization step is about10.0 to about 11.0. More preferably, the pH is about 10.5. The alkalinesolution comprising the dissolved peptide may be allowed to sitquiescently for a period of about 5 minutes to about 3 hours at ambienttemperature, which, although it is not to be construed as a limitation,is generally between about 20° C. and about 25° C. The alkaline solutionmay also be gently stirred. More preferably, the dissolved alkalinepeptide solution will sit quiescently for about 1 hour at ambienttemperature. One skilled in the art will recognize that combinations ofpH, time, temperature and stirring conditions for this step can bereadily established for each peptide that ensures “normalization” of thepeptide conformation is complete yet avoids or minimizes chemicaldegradation that may occur to the peptide.

[0084] The next step in the process for preparing crystals of a selectedpeptide is the addition of glycine. Amino acids such as glycine bindzinc ions which also bind very tightly to the histidine residue(s) in apeptide. Thus, competition for zinc binding may play a role in theformation of peptide crystals, as well as in the stability of subsequentcrystalline compositions. The glycine added to the alkaline peptidesolution may be in a solid form or in a stock solution. Preferably,glycine is added as a solid. Preferably, the added glycine is infree-base form. Preferably, the resulting concentration of glycine inthe alkaline peptide solution is about 5 mM to about 250 mM. Ranges ofmore preferred glycine concentration are about 10 mM to about 150 mM,about 20 mM to about 100 mM, about 40 mM to about 80 mM and about 55 mMto about 65 mM. Most preferably, the glycine concentration is about 60mM.

[0085] Optionally, the pH of the alkaline peptide solution may bereadjusted after the addition of the glycine. If the pH is adjusted, itis preferably adjusted to a pH between about 9.0 and about 11.0. Morepreferably, it is adjusted to a pH between about 9.2 and about 9.8. Mostpreferably, it is adjusted to about pH 9.5.

[0086] Optionally, the alkaline peptide solution with added glycine maybe filtered. Filtration is recommended if any evidence of undissolvedparticles, dust or lint is apparent in the solution. If desired, this isalso a good place in the process at which the solution can be sterilizedby performing an aseptic filtration step. Preferably, the filtrationwill be conducted using a sterile non-pyrogenic filter havinglow-protein binding and a pore size of 0.45 μm or less. Preferably, thefilter is a sterile non-pyrogenic, low-protein binding filter of poresize 0.22 μm or less. More preferably, the filter is a sterile 0.22 μmMillex® filter (Millipore Corporation, Waltham, Mass., USA).

[0087] The next step in the process is addition to the alkaline peptidesolution of about 2% to about 20% of the total final volume of analcohol selected from the group consisting of ethanol and isopropanol,and about 0.2 moles to about 2.5 moles of zinc per mole of the peptide.The zinc and ethanol may be added in a single aqueous stock solution ormay be added separately in one or more steps in any order. Preferably,the alcohol is added before the zinc is added.

[0088] Preferably, the added alcohol represents, by volume, about 2% toabout 20% of the total final volume of the alkaline peptide-zinc-alcoholsolution. More preferably, the alcohol represents about 5% to about 15%of the total final volume. More preferably, the alcohol represents about6% to about 12% of the total final volume. Most preferably, the alcoholrepresents about 9% of the total final volume. Preferably, the alcoholis ethanol.

[0089] The zinc added at this stage refers to the zinc ion. The zinc maybe added in a variety of forms, but a zinc oxide solution acidified withdilute HCl and salt forms such as zinc acetate or zinc chloride arepreferred. More preferred is a zinc oxide solution acidified with diluteHCl.

[0090] Preferably, 1.0 moles to about 2.25 moles of zinc per mole of thepeptide is added in this process step. Other preferred ranges of zincaddition include 1.1 to 2.0 moles of zinc per mole of the peptide, 1.3to 1.7 moles per mole of peptide, and 1.4 to 1.6 moles per mole ofpeptide. Most preferably, about 1.5 moles of zinc per mole of peptide isadded.

[0091] Preferably, the solution comprising zinc that is added to thepeptide solution is added slowly and/or in small increments, whichminimizes the localized precipitation of peptide and/or zinc complexesthat may form at the site of addition. More preferably, glycine is alsoa component of the solution comprising zinc that is being added at thisstep. For example, a zinc-glycine solution may be prepared by dissolvingzinc oxide in dilute HCl to a pH of about 1.6 and then adding solidglycine. A sufficient quantity of glycine is added to raise the pH ofthe solution to between about pH 2 and about pH 3. The pH of thezinc-glycine solution may be raised further using, for example, diluteNaOH. A preferred pH range of the zinc-glycine solution is about pH 4.0to about pH 6.0. A more preferred pH range of the zinc-glycine solutionis about pH 5.0 to about pH 5.5. As noted earlier, glycine has a bindingaffinity for zinc that may compete with zinc binding to the peptide.Thus, the presence of glycine in the solution comprising zinc that isbeing added to the composition allows the zinc solution to be added morequickly because localized precipitation problems are minimized. Inaddition, having a zinc-glycine solution above pH 2.0, and preferablyabout pH 4.0 to about pH 6.0, allows the solution to be sterile filteredusing filters that are rated by their manufacturers to handle, forexample, pH 2-10 solutions, prior to its introduction into a sterilepeptide composition. Preferably, the zinc-glycine solution comprisesabout 50 mM to about 70 mM glycine and about 20 mM to about 200 mM zinc.

[0092] The last steps in the initial crystallization of a selectedpeptide are adjusting the pH of the solution to between about pH 7.5 andabout pH 10.5 and allowing crystals of the peptide to form. Preferredreagent solutions useful for adjusting the pH of the solution includedilute HCl, dilute acetic acid and dilute NaOH.

[0093] Preferred pH ranges for crystallization of selected peptidesinclude about pH 8.0 to about pH 10.0, about pH 7.5 to about pH 9.5,about pH 8.5 to about pH 9.2, about pH 9.0 to about pH 9.5, about pH 7.5to about pH 8.5, about pH 8.7 to about pH 9.5, and about pH 9.2 to aboutpH 10.0.

[0094] One skilled in the art will recognize that the preferred pH ofcrystallization will depend on many factors, including the concentrationof peptide, the alcohol concentration, the zinc concentration, the ionicstrength of the solution and the temperature of crystallization.

[0095] The skilled artisan will further recognize that, for a given setof conditions, a preferred manner of determining the optimal pH ofcrystallization is to determine it empirically, that is, to slowly addthe acidification solution, preferably dilute HCl or dilute acetic acid,in small increments, and observe what happens after each increment isadded. Generally, small quantities of localized zones of precipitationwill occur at the spot of addition of the acidic solution. When gentleswirling takes increasingly longer periods of time to completelyredissolve the precipitation, that is the best time to stop adding theacid and allow crystallization from the clear or slightly cloudysolution to proceed.

[0096] The skilled artisan will further recognize that the pH andtemperature that one selects for crystallization will have an impact onthe speed at which the crystallization proceeds, the crystallizationyield, and the size and homogeneity of the crystals formed. Preferably,the pH of crystallization for the selected peptides is about pH 8.0 toabout pH 10. More preferably, the pH is about 8.7 to about 9.5. Otherranges of preferred pH of crystallization are about 8.8 to about 9.3,about 9.0 to about 9.5, and about 8.5 to about 9.3. Most preferably, thecrystallization is conducted at about pH 9.1.

[0097] Preferably, the temperature of crystallization is about 10° C. toabout 30° C. More preferably, the temperature of crystallization isabout 15° C. to about 28° C. Most preferably, the temperature ofcrystallization is ambient temperature, or about 20° C. to about 25° C.

[0098] Preferably, the crystallization step described above is complete,that is, 90% or more of the peptide is precipitated in predominantlycrystalline form, in about 3 hours to about 72 hours. More preferably,the crystallization is complete in about 10 hours to about 48 hours.Most preferably, the crystallization is complete in about 16 hours toabout 26 hours. Completion of crystallization may be determined by avariety of means, including HPLC analysis of the peptide present in analiquot of the composition. Method 5 herein describes one such protocolthat may be employed.

[0099] Preferably, the crystals produced according to the steps of theprocess described above are thin plate crystals. The crystals producedby the process may be examined by microscopy.

[0100] Pharmaceutical” compositions comprising crystals of a peptideprepared as described above may be prepared by adding suitable,pharmaceutically acceptable excipients to the crystal suspension in theoriginal mother liquor. Alternatively, the crystals may be isolated byfiltration, gentle centrifugation or other means of phase separation,and used in a variety of ways to prepare pharmaceutically acceptablecompositions. The skilled artisan will recognize suitable procedures andexcipients useful for preparing such pharmaceutical compositions.

[0101] To prepare a stable pharmaceutical composition of crystals of aselected peptide, the pH of the suspension of crystals in their completeoriginal mother liquor, or portion thereof, is lowered to a pH value atwhich 97% or more of the peptide becomes insoluble. Preferably, thispart of the process begins within a few hours after the initialcrystallization is determined to be complete. Preferably, the pH islowered using a dilute solution of HCl or acetic acid wherein the acidicsolution is added slowly and in incremental portions. The skilledartisan will recognize that the preferred pH at which this second stageof crystallization should occur will depend on many factors, includingthe nature of the peptide and its concentration, the alcoholconcentration, the zinc concentration, the ionic strength of thesuspension and the temperature of crystallization. Preferably, the pH isabout 0.2 to 2.0 pH units lower than the pH at which the initialcrystallization proceeded. More preferably, the pH is about 0.5 to about1.5 pH units lower, and most preferably, the pH is about 0.8 to 1.3 pHunits lower than the pH at which the initial crystallization proceeded.The temperature of this second stage of crystallization is preferablyambient temperature, or about 20° C. to about 25° C. For the peptideVal⁸-GLP-1(7-37)OH, a preferred pH is about 7.5 to about 8.5. A morepreferred pH is about 7.8 to about 8.2.

[0102] Preferably, the pH of a suspension of peptide crystals is loweredto a pH at which 98% or more, and more preferably at which 99% or moreof the peptide becomes insoluble in the composition. The additionalprecipitation formed in this second stage of crystallization comprisescrystals. Preferably, the additional precipitation formed in this secondstage of crystallization will be predominantly crystals of comparablemorphology and size distribution as those formed in the first stage ofcrystallization.

[0103] Preferably, the second stage of crystallization is completeenough, that is, 97% or more of the peptide is insoluble, to allow thefollowing step to begin within 30 hours, more preferably within 18hours, more preferably within 6 hours and most preferably within 2 hoursof when the second stage of crystallization started. Quantitation ofprecipitation yield may be determined by a variety of means, includingHPLC analysis of the peptide present in an aliquot of the composition.Method 5 herein describes one such protocol that may be employed.

[0104] The next step in the process to prepare a stable pharmaceuticalcomposition of crystals of a selected peptide is to add apharmaceutically acceptable preservative and buffer selected from thegroup consisting of TRIS and maleate. Optionally, one or more tonicityagents such as sodium chloride, other salts, glycerin or mannitol mayalso be added. These components may be added as a single solution, ascombination solutions or individually in any order. It is preferred thatthe preservative is added last. Of these components, a preferred bufferis selected from the group consisting of TRIS and maleate, a preferredpreservative is m-cresol and a preferred tonicity agent is sodiumchloride. A more preferred buffer is TRIS.

[0105] A preferred quantity of TRIS to add to the crystalline peptidesuspension, if TRIS is the selected buffer, is such that the TRISconcentration in the final composition is about 5 mM to about 40 mM. Amore preferred range of TRIS concentration in the final composition isabout 10 mM to about 20 mM. A most preferred concentration of TRIS inthe final composition is about 15 mM.

[0106] A preferred quantity of maleate to add to the crystalline peptidesuspension, if maleate is the selected buffer, is such that the maleateconcentration in the final composition is about 2 mM to about 20 mM. Amore preferred range of maleate concentration in the final compositionis about 5 mM to about 15 mM. A most preferred concentration of maleatein the final composition is about 9 mM.

[0107] If sodium chloride is selected to be a component of a peptidecomposition of the present invention, a preferred quantity to add to thecrystalline peptide suspension is such that the added sodium chloride inthe final composition is about 30 mM to about 200 mM. A more preferredconcentration of added sodium chloride in the final composition is 50 mMto about 150 mM. Other ranges of preferred sodium chloride concentrationare about 80 mM to about 120 mM, about 70 mM to about 130 mM, and about90 mM to about 130 mM. A most preferred quantity of added sodiumchloride in a pharmaceutical composition of the present invention isabout 110 mM.

[0108] Although any pharmaceutically acceptable preservative may beadded to the crystalline peptide suspension at this point in theprocess, for a composition of the present invention a phenolicpreservative or benzyl alcohol is preferred. Examples of phenolicpreservatives include phenol, chlorocresol, m-cresol, o-cresol,p-cresol, ethylparaben, methylparaben, propylparaben, butylparaben,thymol or mixtures thereof. More preferred preservatives are benzylalcohol, m-cresol, phenol, methylparaben and mixtures thereof. A mostpreferred pharmaceutically acceptable preservative is m-cresol.

[0109] A preferred quantity of a pharmaceutically acceptablepreservative to add to a crystalline peptide composition at this pointin the process is an amount such that the preservative concentration inthe final composition is about 1.0 mg/mL to about 20.0 mg/mL. Morepreferred ranges of concentration of preservative in the finalcomposition are about 2.0 mg/mL to about 8.0 mg/mL, about 2.5 mg/mL toabout 4.5 mg/mL and about 2.0 mg/mL to about 4.0 mg/mL. A most preferredconcentration of preservative in the final composition is about 3.0mg/mL.

[0110] The final step in the process of preparing a stablepharmaceutical composition of crystals of a selected peptide is anadjustment to a final pH between about 6.0 and about 8.5, and preferablybetween about pH 6.5 and about pH 8.0, and more preferably between aboutpH 7.0 and about pH 8.0. Although any of a wide variety of acidificationand/or alkalization reagent solutions may be employed for this pHadjustment, dilute HCl, dilute NaOH and dilute acetic acid arepreferred. More preferred reagent solutions are dilute HCl and diluteNaOH. The preferred pH to which the composition is adjusted will dependto some extent upon the selected peptide, the peptide concentration, theproposed route of administration and the selected buffer.

[0111] Preferably, with TRIS as the selected buffer, the pH will beadjusted to a pH between about 6.5 and about 8.5. More preferably, thepH will be adjusted to a pH between about 7.0 and about 7.8, betweenabout 7.2 and about 7.8, between about 7.5 and about 8.5, or betweenabout 7.0 and about 8.0. A most preferred pH to which the composition isadjusted when TRIS is the selected buffer is about 7.5. With maleate asthe selected buffer, the pH will be adjusted to a pH between about 6.0and about 7.5. More preferably, the pH will be adjusted to a pH betweenabout 6.4 and about 7.5, between about 6.4 and about 7.0, or betweenabout 6.0 and about 7.0. A most preferred pH to which the composition isadjusted when maleate is the selected buffer is about 6.5.

[0112] Another aspect of the present invention is a stablepharmaceutical composition prepared by the process described in thepreceding paragraphs.

Method 1 Sedimentation Rate

[0113] Cartridges or vials containing a suspension of crystals areevaluated visually for crystal settling rate and volume. Afterthoroughly suspending the insoluble material in the cartridges or vials,each composition is allowed to settle quiescently at ambient temperature(about 20° C. to about 25° C.). The resulting sedimentation volume ofthe suspended material in each of the compositions after 1 hour isdetermined as a percentage of total volume. Further, after sittingquiescently for 24 hours at ambient temperature, the sedimentationvolume for each sample is determined as a percentage of total volume.

Method 2 TCR Test

[0114] The TCR (temperature cycling and resuspension) Test is anautomated procedure combining temperature cycling from 25° C. to 37° C.and mechanical agitation that evaluates, formulations under conditionsmore extreme than would be expected for patient usage. This test wasdescribed for insulin suspensions and solutions by Shnek, D. R. et al.,in J. Pharmaceutical Sciences 87:1459-1465 (1998).

[0115] Briefly, the TCR Test employs temperature cycling between 25° C.and 37° C. in an incubator unit combined with resuspensions conductedtwice daily on a mechanical device outside the incubator unit. The 3-mLglass cartridges containing a 1 mm glass bead and rubber plunger arefilled with peptide suspensions and capped with a disk seal. The samplesare held in a horizontal position for 10 hours per day at 25° C. and 10hours per day at 37° C., with 2-hour temperature ramping steps betweenthem. The mechanical resuspension of the test cartridges performs threesets of 10 lateral rolls plus 10 cartridge inversions twice daily. Theresuspensions are conducted during the 25° C. temperature cycle. Theentire test lasts up to 28 days. For additional details of the TCR Testsee Shnek, D. R. et al., supra.

Method 3 Modified TCR Test

[0116] The Modified TCR Test is similar to the TCR Test described inMethod 1 except that the temperature cycles are 5° C. and 25° C. Theresuspensions are conducted during the 25° C. cycle.

Method 4 Particle Size Measurements

[0117] Particle size measurements are performed using a Coulter ModelLS230 Particle Size Analyzer (Beckman Coulter, Inc., Fullerton, Calif.,USA). An aliquot (about 100 μL) of the test suspension is diluted about100-fold to about 300-fold by pipetting it into a diluent containing 2.4mM zinc, 150 mM NaCl and 50 mM sodium acetate at pH 5. Particle sizedata are collected for about 120 seconds, and the resulting distributionis analyzed with Coulter particle size statistics software which assumeda spherical shape. The mean volume percent distribution of particlesizes is obtained and reported.

Method 5 Visual Assessment of Peptide Compositions

[0118] Cartridges of peptide suspension compositions being evaluated inthe TCR Test and in the Modified TCR Test are examined by trainedoperators on various test days. The test compositions are checked forvisual changes compared to quiescently refrigerated control samples.Visual changes included the presence of large aggregates (alsodesignated as clumps) and/or material that adheres to the cartridge wall(also designated as frosting). For the evaluation of test results, amilky white suspension or the presence of grainy material thatresuspends upon swirling is reported as PASS while visual changesinvolving large-aggregates, clumps, frosting, or grainy material thatdoes not resuspend upon swirling is reported as FAILED. Additionaldetails of these evaluation criteria are described in Shnek, D. R. etal., supra.

Method 6 HPLC Analysis of Peptides

[0119] Peptides in the aqueous compositions of the present invention areanalyzed on a 25 cm×4.6 mm Zorbax 300 SB (C-8) HPLC column (Mac-ModAnalytical Inc., Chadds Ford, Pa., USA) with UV detection at 214 nm. Agradient made from a first solution (0.1% TFA in water) and a secondsolution (0.1% TFA in acetonitrile) is used to effect elution of thepeptides and related impurities.

[0120] Method 5 is used to determine the purity of peptides insuspensions undergoing the TCR Test (Method 2) and the Modified TCR Test(Method 3). For these analyses, an aliquot of the suspended compositionis diluted about 10-fold or greater with a 2 M guanidine-0.01 N HClsolution to solubilize the peptide crystals prior to injection.

[0121] To quantitate the soluble peptide present in a suspensioncomposition, portions of the swirled suspension are centrifuged and analiquot of the clear supernatant is analyzed by HPLC as described above.

Method 7 FTIR Analysis

[0122] FTIR (Fourier Transform Infrared Raman) spectra are acquired on aNicolet Nic-Plan FTIR microscope using a Nicolet 760 Spectrometeroptical bench (Nicolet Instrument Corporation, Madison, Wis., USA) andOmnic version 5.1 software. Microliter portions of the peptide samplesare placed on a glass slide and allowed to evaporate to dryness in adesiccator overnight at ambient temperature (20° C. to 25° C.). Thedried peptide is removed from the slide using a micro-tool and placed ona diamond anvil cell (Spectra-Tech, Inc., Shelton, Conn., USA) foranalysis. Spectra were acquired at 4 cm⁻¹ resolution from 128 scans andare baseline adjusted. The background spectra are similarly obtainedwith half of the diamond anvil cell in the beam. For analysis ofsecondary structure, second derivatives and deconvolved spectra areobtained using Nicolet software. Second derivatives are inverted(multiplied by −1000) and smoothed (11 points over 10.6 cm⁻¹).Deconvolved spectra are obtained using a 23 cm⁻¹ bandwidth and 2.2enhancement factor.

[0123] The following examples are provided merely to further illustratethe invention. The scope of the invention shall not be construed asmerely consisting of the following examples.

EXAMPLE 1 Chemical Synthesis of Val⁸-GLP-1(7-37)NH₂

[0124] Peptides are prepared by the solid-phase method of Merrifield etal. (1963) J. Am. Chem. Soc. 85:2149-2154 either by the originalt-Butyloxycarbonyl/Benzyl (t-Boc/Bzl) methodology orfluroenyloxycarbonyl/t-Butyl (Fmoc/tBu) based method.

[0125] t-Boc/Bzl Synthesis:

[0126] The synthesis is initiated with 4-methylbenzhydrylamine (4-MBHA)derivatized polystyrene resin which is used to anchor the 1^(st)residue, t-Boc protected glycinamide. The synthesis can be carried outmanually (on variable scale) or by automated stepwise fashion using apeptide synthesizer (typically 0.25 mmole scale). Boc group deprotectionis carried out by treatment with trifluoroacetic acid, the freeN-terminus is neutralized with diisopropylethylamine either prior to orsimultaneously with the next coupling. Boc protected amino acids areactivated with DIC (diisopropylcarbodiimide) or DCC(dicyclohexylcarbodiimide) to form symmetrical anhydrides or with HBTU(hydroxybenztriazolyl tetramethyluronium hexafluorophosphate) to formactivated esters. This deprotection-neutralization-coupling cycle isrepeated until the peptide sequence is fully assembled. Conventionalside chain protection is as follows: Arg(Tosyl), Asp(cHexyl)Gln(Xanthyl), Glu(cHexyl) His(Boc), Lys(2-ClBenzyl), Ser(Benzyl), Thr(Benzyl), Trp(formyl), Tyr(2-BrBenzyl). The protected peptide issimultaneously cleaved from the support and deprotected using liquidhydrogen fluoride containing an appropriate scavenger such as cresol orm-cresol. The peptide is precipitated with diethyl ether, purified byhigh performance liquid chromatography and lyophilized.

[0127] Fmoc/tBu Synthesis:

[0128] The synthesis is initiated with 2,4-dimethoxy-benzhydrylamine(“Rink” type) polystyrene resin which is used to anchor the 1^(st)residue, Fmoc protected glycinamide. Again, the synthesis can be carriedout manually (on variable scale) or by automated stepwise fashion usinga peptide synthesizer (typically 0.25 mmole scale). Fmoc groupdeprotection is carried out by treatment with piperidine indimethylformamide. Fmoc protected amino acids are activated with DIC(diisopropylcarbodiimide) or DCC (dicyclohexylcarbodiimide) to formsymmetrical anhydrides or with HBTU (hydroxybenztriazolyltetramethyluronium hexafluorophosphate) to form activated esters. Thisdeprotection-coupling cycle is repeated until the peptide sequence isfully assembled. Conventional side chain protection is as follows:Arg(pentamethylbenzofuran), Asp(O-tButyl), Gln(trityl), Glu(O-tButyl)His(t-Butyloxycarbonyl), Lys(t-Butyloxycarbonyl), Ser(O-tButyl),Thr(O-tButyl), Trp(t-butyloxycarbonyl), Tyr(O-tButyl). The protectedpeptide is simultaneously cleaved from the support and deprotected usingtrifluoroacetic acid containing an appropriate scavenger such asethanedithiol and water. The cleavage mixture is concentrated, thepeptide precipitated with diethyl ether. The crude peptide is purifiedby high performance liquid chromatography and lyophilized.

EXAMPLE 2 Trypsin Mediated Transpeptidation Val⁸GLP-1(7-37)OH toVal⁸-GLP-1(7-37)NH₂

[0129] Val⁸-GLP-1(7-37)OH, is converted to the Val⁸-GLP-1(7-37)NH2 usingtrypsin mediated transpeptidation to remove the C-terminal glycineresidue and replace with a glycinamide residue. Trypsin is used becauseof its selectivity for basic amino acid residues (lysine, arginine) andthat the penultimate amino acid residue at the C-terminal end ofVal⁸-GLP-1(7-37)OH is arginine. To prevent trypsin digesting theVal⁸-GLP(7-37)OH at each of the two lysine the Val⁸-GLP-1(7-37)OH istreated with an excess of citriconic anhydride in an aqueous bufferedsolution (pH 9-10) to temporarily protect the lysine residues and renderthem “invisible” to trypsin. This also caps the N-terminal amino groupof Val⁸-GLP-1(7-37)OH. Next, the excess citriconic anhydride is quenchedwith ethanolamine. Then trypsin is added (1:10 vs. Val⁸GLP-1(7-37)OH ona weight basis) from its stock solution (50 mM calcium acetate in water)under constant stirring (pH 8-9). This converts the Val⁸-GLP-1(7-37)OHto the Val⁸-GLP-1(7-36)OH by removing the C-terminal glycine residue.After thirty minutes of digestion a 50 fold molar excess of glycinamidehydrochloride suspended in dimethylacetamide (volume calculated toresult in a 35/65% (v/v) aqueous/organic solution (pH 8-9)). Thereaction mixture is then placed on a stir plate in a refrigerator at 4degrees centigrade and the conversion monitored by analyticalreversed-phase HPLC and mass spectra.

[0130] Removal of citriconyl groups for HPLC analysis is effected byincubating a 10-fold diluted sample (in 0.1 M H3PO4/6M GdnHCl) at 110degrees centigrade for 10 minutes.

EXAMPLE 3 Aggregation Time Val⁸-GLP-1(7-37)OH and Val⁸-GLP-1(7-37)NH₂

[0131] Val⁸-GLP-1(7-37)OH and Val⁸-GLP-1(7-37)NH₂ were analyzed withrespect to their potential to aggregate in solution. In general, thepeptides in solution were stirred at elevated temperature in a suitablebuffer while recording turbidity at 350 nm as a function of time. Timeto the onset of aggregation was measured to quantify the potential of agiven GLP molecule to aggregate under these stressed conditions.

[0132] The GLP-1 peptides were first dissolved under alkaline conditions(pH-10.5) for 30 minutes to dissolve any pre-aggregated material. Thesolution was then adjusted to pH 7.4 and filtered. Specifically, 4 mg ofa lyophilized GLP-1 peptide was dissolved in 3 mL of 10 mM phosphate/10mM citrate. The pH was adjusted to 10.0-10.5 and held for 30 minutes.The solution was adjusted with HCl to pH 7.4 and filtered through asuitable filter, for example a Millex® GV syringe filter (MilliporeCorporation, Bedford, Mass.). This solution was then diluted to a finalsample containing 0.3 mg/mL protein in 10 mM citrate, 10 mM phosphate,150 mM NaCl, and adjusted to pH 7.4 to 7.5. The sample was incubated at37° C. in a quartz cuvette. Every five minutes the turbidity of thesolution was measured at 350 nm on an AVIV Model 14DS UV-VISspectrophotometer (Lakewood, N.J.). For 30 seconds prior to and duringthe measurement the solution was stirred using a magnetic stir bar fromStarna Cells, Inc. (Atascadero, Calif.). An increase in OD at 350 nmindicates aggregation of the GLP-peptide. The time to aggregation wasapproximated by the intersection of linear fits to the pre-growth andgrowth phase according to method of Drake (Arvinte T, Cudd A, and DrakeA F. (1993) J. Bio. Chem. 268, 6415-6422)

[0133] The cuvette was cleaned between experiments with a caustic soapsolution (e.g., Contrad-70).

[0134] Val⁸-GLP-1(7-37)OH aggregated in approximately 1.5 hours at 30°C. and approximately 1 hour at 37° C. Val⁸-GLP-1(7-37)NH₂ showed asubstantially reduced tendency to aggregate. Val⁸-GLP-1(7-37)NH₂ did notaggregate for approximately 23 hours at 37° C.

EXAMPLE 4 In Vitro Activity Val⁸-GLP-1(7-37)NH₂ relative toVal⁸-GLP-1(7-37)OH

[0135] HEK-293 Aurora CRE-BLAM cells expressing the human GLP-1 receptorare seeded at 20,000 to 40,000 cells/well/100 μl into a 96 well blackclear bottom plate. The day after seeding, the medium is replaced withplasma free medium. On the third day after seeding, 20 μL of plasma freemedium containing different concentrations of GLP-1 peptide is added toeach well to generate a dose response curve. Generally, fourteendilutions containing from 3 nm to 30 nm GLP-1 peptide were used togenerate a dose response curve from which EC50 values could bedetermined. After 5 hours of incubation with GLP-1 peptide, 20 μL ofβ-lactamase substrate (CCF2-AM—Aurora Biosciences—product code 100012)was added and incubation continued for 1 hour at which point thefluorescence was determined on a cytofluor. The in vitro activity ofVal⁸-GLP-1(7-37)NH₂ relative to the in vitro activity ofVal⁸-GLP-1(7-37)OH for 7 different samples is illustrated in Table 2below. TABLE 2 Val⁸-GLP-1(7-37)NH₂ Sample # (% Val⁸-GLP-1(7-37)OH) 1 1502 106 3 128 4 125 5 133 6 92 7 79

EXAMPLE 5 Crystallization of Val⁸-GLP-1(7-37)NH2

[0136] Val⁸-GLP-1(7-37)NH₂ was dissolved in about 0.48 mL of 0.015 NNaOH at a concentration of about 17 mg/mL, based on the mass of thepeptide. The protein solution was adjusted to about pH 11.1 with diluteNaOH, then to pH 10.36 with dilute HCl. The solution was held at ambienttemperature for about 1 hour.

[0137] To a 390 μL aliquot of this peptide solution was added 25 μL of a1.0 M glycine pH 10 solution, giving a final concentration of about 16mg/mL of Val⁸-GLP-1(7-37)NH₂ and about 60 mM glycine.

[0138] The solution was then filtered into a glass vial through asterile 0.22 μm Millex®-GV (Millipore Corporation, Waltham, Mass., USA)4 mm filter unit.

[0139] To 300 μL of the filtered peptide solution in a clean glass vialwas added 66 μL of a 50% ethanol solution in water. To this solution wasadded, in small increments, a total of about 7 μL of a 150 mM zinc oxidepH 2.3 solution (prepared with dilute HCl), with mixing by handperformed after each increment was added until the solution becameclear. The molar ratio of zinc:peptide was about 0.75:1.

[0140] The final solution was adjusted to about pH 9.8 andcrystallization proceeded at ambient temperature. The crystallizationsolution comprised about 12.6 mg/mL Val⁸-GLP-1(7-37)NH₂, 47 mM glycine,8.7% ethanol by volume, and about 0.75 moles of zinc per mole ofVal⁸-GLP-1(7-37)NH₂ at pH 9.8.

[0141] After about 48 hours at ambient temperature, clusters ofVal⁸-GLP-1(7-37)NH₂ were observed under a microscope at 400×magnification.

1 3 1 31 PRT Artificial Sequence synthetic construct 1 His Xaa Glu GlyThr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa 1 5 10 15 Gln Ala AlaLys Xaa Phe Ile Xaa Trp Leu Val Lys Gly Arg Xaa 20 25 30 2 31 PRT Homosapiens 2 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu GluGly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly ArgGly 20 25 30 3 31 PRT Artificial Sequence Synthetic construct 3 His ValGlu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 GlnAla Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30

We claim:
 1. A peptide comprising the sequence of Formula I (SEQ ID NO:1): Formula I 7   8   9   10  11  12  13  14  15  16  17 (SEQ ID NO: 1)His-Xaa-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-18  19  20  21  22  23  24  25  26  27  28Ser-Tyr-Leu-Glu-Xaa-Gln-Ala-Ala-Lys-Xaa-Phe-29  30  31  32  33  34  35  36  37 Ile-Xaa-Trp-Leu-Val-Lys-Gly-Arg-R

wherein: Xaa at position 8 is Val; Xaa at position 22 is Gly; Xaa atposition 27 is Glu; Xaa at position 30 is Ala; and R is Gly-NH₂. 2.Crystals of the peptide of claim
 1. 3. A pharmaceutical compositioncomprising the crystals of claim
 2. 4. The pharmaceutical composition ofclaim 3 further comprising: zinc, a buffer, and apharmaceutically-acceptable preservative.
 5. The pharmaceuticalcomposition of claim 4 further comprising glycine and an alcoholselected from the group consisting of ethanol and isopropanol.
 6. Thepharmaceutical composition of claim 5 wherein the buffer is selectedfrom the group consisting of TRIS and maleate and the pH is betweenabout 6.0 and about 8.5.
 7. The pharmaceutical composition of claim 6wherein the zinc is present at a concentration of about 0.2 moles toabout 2.5 moles per mole of the peptide.
 8. The pharmaceuticalcomposition of claim 7 wherein the glycine is present at a concentrationof about 5 mM to about 100 mM and the alcohol is present at aconcentration by volume of about 1% to about 10%.
 9. The pharmaceuticalcomposition according to any one of claims 3 to 8, further comprising atonicity agent.
 10. The pharmaceutical composition according to claim 9,wherein the tonicity agent is glycerin.
 11. The pharmaceuticalcomposition according to claim 9, wherein the tonicity agent is sodiumchloride.
 12. The pharmaceutical composition according to claim 11,wherein the sodium chloride is at a concentration of about 50 mM toabout 150 mM.
 13. The pharmaceutical composition according to any one ofclaims 5 to 12, wherein the glycine concentration is about 10 mM toabout 50 mM.
 14. The pharmaceutical composition according to claim 13,wherein the glycine concentration is about 20 mM to about 30 mM.
 15. Thepharmaceutical composition according to any one of claims 5 to 14,wherein the alcohol concentration is about 2% to about 6% by volume. 16.The pharmaceutical composition according to any one of claims 5 to 15,wherein the alcohol is ethanol.
 17. The pharmaceutical compositionaccording to any one of claims 4 to 16, wherein the zinc concentrationis about 1.1 to about 2.0 moles per mole of the peptide.
 18. Thepharmaceutical composition according to any one of claims 4 to 17,wherein the buffer is TRIS.
 19. The pharmaceutical composition accordingto any one of claims 4 to 18, wherein the preservative is a phenolicpreservative.
 20. The pharmaceutical composition according to claim 19,wherein the preservative is m-cresol.
 21. The pharmaceutical compositionaccording to any one of claims 3 to 20, wherein the peptideconcentration is about 2.0 mg/mL to about 30.0 mg/mL.
 22. Thepharmaceutical composition according to claim 21, wherein the peptideconcentration is about 2.0 mg/mL to about 8.0 mg/mL.
 23. Apharmaceutical solution formulation comprising the peptide of claim 1.24. The pharmaceutical solution formulation of claim 23 furthercomprising a buffer.
 25. The pharmaceutical solution formulation ofclaim 24 wherein the formulation has a pH between about 6.0 and about8.0.
 26. A lyophilized formulation comprising the peptide of claim 1.27. The lyophilized formulation of claim 26 further comprising a bulkingagent selected from the group consisting of mannitol., trehalose,raffinose, and sucrose.
 28. The lyophilized formulation of claim 27wherein the bulking agent is mannitol.
 29. The lyophilized formulationof any one of claims 26 to 28 further comprising a buffer system suchthat upon reconstitution the resulting formulation has a pH betweenabout 6.0 and about 8.0.
 30. Use of the pharmaceutical compositionaccording to any one of claims 3 to 22 in the manufacture of amedicament for treating type 2 diabetes, hyperglycemia, or obesity in amammal.
 31. Use of the solution formulation according to any one ofclaims 23 to 25 for the manufacture of a medicament to reduce themorbidity and mortality associated with myocardial infarction or stroke.32. Use of solution formulation according to any one of claims 23 to 25for the manufacture of a medicament to attenuate catabolic changes thatoccur after surgery.
 33. Use of the lyophilized formulation according toany one of claims 26 to 29 for the manufacture of a medicament to reducethe morbidity and mortality associated with myocardial infarction orstroke.
 34. Use of the lyophilized formulation according to any one ofclaims 26 to 29 for the manufacture of a medicament to attenuatecatabolic changes that occur after surgery.
 35. A pharmaceuticalcomposition according to any one of claims 3 to 22 for the treatment ofa human or animal body by therapy.
 36. A formulation according to anyone of claims 23 to 29 for the treatment of a human or animal body bytherapy.
 37. The peptide of claim 1 when made by the process oftranspeptidation.
 38. A process of modifying the peptide of claim 1comprising the steps: a) preparing an aqueous solution comprisingGLP-1(7-37)OH; b) adding an enzyme that adds protecting groups to thelysine residues in GLP-1(7-37)OH to prevent trypsin from cleaving afterthe lysine residues; c) digesting GLP-1(7-37)OH with trypsin; d) addinga molar excess of glycinamide hydrochloride; and e) removing theprotecting groups from the lysine residues.
 39. The process of claim 38wherein the enzyme of step (b) is citriconic anhydride.
 40. The processof claim 39 further comprising quenching the reaction with ethanolaminebefore adding trypsin.